TW202334433A - Virus-like particle stably expressed by animal cells as vaccine antigen against covid-19 and influenza virus - Google Patents

Abstract

The disclosure provides an animal cell stably expressing a virus-like particle (VLP). The disclosure also provides a method for manufacturing a virus-like particle, a virus-like particle, a vaccine composition, a method for preventing viral infection, and a method for producing antibodies.

Description

Using virus particles stably expressed in animal cells as vaccine antigens against COVID-19 and influenza viruses

The present invention relates to vaccines. More specifically, the present invention relates to virus-like particles stably expressed by animal cells.

The use of proteins as therapeutics has been approved by the US FDA and is expected to grow significantly. However, the manufacture of protein drugs relies on high gene expression. For the production of proteins with extensive post-translational modifications, mammalian and insect cell lines appear to be the best or most acceptable expression systems. In addition, the ability to produce vaccines to quickly respond to emerging infectious diseases, such as COVID-19, 2009 H1N1 new influenza... and other pandemics, is critical to controlling the outbreak of infectious diseases.

An ideal vaccine must be highly immunogenic and confer elimination immunity and infection protection within a safe range. Therefore, advanced vaccines should include structurally engineered antigens that present neutralizing epitopes with a stable structure on the surface; a delivery system that promotes uptake of the vaccine by antigen-presenting cells; and adjuvant activity that interacts with immune receptors. It binds to the body and triggers a protective immune response ( Koff et al. , Sci Transl Med 13 , 2021 ). In addition to the newly developed genetic vaccines currently in use, such as lipid nanoparticle-coated message ribonucleic acid (mRNA-LNP) and adenovirus vectors carrying antigen encoding, the conventional inactivated virus-based vaccines and protein vaccines are also in use. Under development ( Keech et al. , N Engl J Med 383, 2320-2332, 2020 ; Richmond et al. , Lancet 397, 682-694, 2021 ; Kuo et al. , Sci Rep 10, 20085, 2020 ; Krammer, Nature 586, 516 -527, 2020 ). Virus-based vaccines are not yet fully available in developed countries due to the high risk of large-scale viral culture and incomplete deactivation, as well as the threat of pre-existing cross-immune responses to previously exposed common cold coronaviruses. Protein vaccines generally have low immunogenicity and require appropriate adjuvants to activate immune responses. Virus-like particles (VLPs) and nanoparticle platforms reproduce the particle characteristics of viruses and enhance the delivery of antigens to lymphoid tissues and facilitate uptake by dendritic cells. They are also promising in vaccine research.

However, multiple genes must be rapidly co-expressed in cell lines to achieve rapid, stable and large-scale production.

The present invention relates to a performance system for rapid, stable and large-scale production of viral vaccines.

The present invention provides an animal cell that stably expresses virus-like particles, which contains an inducible expression cassette for site-directed recombination embedded with one or more VLP genes.

The animal cells may be human cells. Examples of animal cells include, but are not limited to, insect or mammalian cells. Mammalian cells may be derived from rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, pigs (such as miniature pigs), cattle, horses, primates (such as monkeys, including macaques), or humans. Insect cells can be exemplified by Lepidoptera, and examples thereof include but are not limited to Spodoptera frugiperda , Bombyx mori , Heliothis virescens , Heliothis zea , Mamestra brassicas ), Estigmene acrea and Trichoplusia ni .

In some embodiments of the invention, the virus is an enveloped virus. Examples of enveloped viruses include, but are not limited to, sindbis virus, rubella virus, yellow fever virus, hepatitis C virus, influenza virus, measles virus, mumps virus, human metapneumovirus, respiratory fusion virus , vesicular stomatitis virus, rabies virus, Hantan virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, coronavirus, SARS virus, lymphocytic Choriomeningitis virus (LCM virus), human T-cell leukemia virus, human immunodeficiency virus (HIV), Marburg virus, Ebola virus, human herpes virus, pox virus and hepatitis B virus. In particular, the virus is a coronavirus or an influenza virus. In one embodiment of the invention, the virus is SARS-CoV-2 (COVID-19) or H5N2 or H3N2 influenza virus.

In some embodiments of the invention, the virus-like particles comprise coronavirus structural proteins or influenza virus structural proteins.

In one embodiment of the present invention, the coronavirus structural protein is the structural protein of SARS-CoV-2. In some embodiments of the invention, SARS-CoV-2 is a Delta and Omicron variant of SARS-CoV-2. Coronavirus structural proteins as disclosed herein include spike protein (S), membrane protein (M) and envelope protein (E). Examples of spike proteins include, but are not limited to, native D614G spike protein (SEQ ID NO: 6), diproline mutant spike protein (2P-S) (SEQ ID NO: 8), D614G-S mutant spike protein , Delta-or spike protein (SEQ ID NO: 10), Delta-GSAS-2P spike protein (SEQ ID NO: 12), Omicron-or spike protein (SEQ ID NO: 14), Omicron-GSAS-2P Spike protein (SEQ ID NO: 16). In one embodiment of the present invention, the coronavirus structural protein is encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 7, 9, 11, 13, 15 and 17.

In one embodiment of the present invention, the influenza virus structural protein is the structural protein of H5N2 influenza virus (eg, A/duck/Taiwan/01006/2015/H5N2). Influenza virus structural proteins as disclosed herein include hemagglutinin (HA), neuraminidase (NA) and matrix proteins (M1 and M2). In some embodiments of the invention, the H5 protein is as shown in SEQ ID NO: 18, and the codons of the H5 protein are optimized as shown in SEQ ID NO: 19. In some embodiments of the invention, the N2 protein is as shown in SEQ ID NO: 20, and the codons of the N2 protein are optimized as shown in SEQ ID NO: 21. In one embodiment of the present invention, the influenza virus structural protein is the structural protein of H3N2 influenza virus (eg, A/Taiwan/083/2006/H3N2). In some embodiments of the invention, the M1 protein is as shown in SEQ ID NO: 22, and the codons of the M1 protein are optimized as shown in SEQ ID NO: 23. In some embodiments of the invention, the M2 protein is as shown in SEQ ID NO: 24, and the codons of the M1 protein are optimized as shown in SEQ ID NO: 25.

In one embodiment of the present invention, the animal cell line is stably transfected with a target cassette containing an inducible tetracycline-inducible promoter-reporter or a doxycycline-inducible promoter-reporter of the Flp/FRT recombinant system. and a stably expressed tetracycline repressor cassette; and was established by gene exchange of the target cassette and the tetracycline-inducible promoter-reporter cassette by co-transfection with FLPe recombinase to achieve site-specific insertion of all VLP genes.

In some embodiments of the invention, the inducible expression cassette comprises a tetracycline-inducible promoter or a doxycycline-inducible promoter. An example of a tetracycline-inducible promoter is the CMV/TO promoter. Examples of doxycycline-inducible promoters include, but are not limited to, Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) immediate early 2 (IE2) and Antheraea pernyl actin A1 promoter.

In some embodiments of the invention, animal cells also stably express the tetracycline inhibitor cassette.

In some embodiments of the invention, the tetracycline suppressor cassette includes a tetracycline suppressor gene and a puminomycin S resistance gene linked by a self-cleaving 2A peptide derived from porcine Tieskovirus 1.

In some embodiments of the invention, the tetracycline inhibitor cassette is an EF1a/eIF4g-pCI-TetR-P2A-BSR cassette.

The invention also provides a method for producing virus-like particles, comprising culturing animal cells as described herein and harvesting virus-like particles.

The invention also provides a virus-like particle produced by a method comprising culturing animal cells as described herein and harvesting the virus-like particle.

The present invention provides a vaccine composition comprising an immunologically effective amount of virus-like particles produced by a method comprising culturing animal cells as described herein and harvesting virus-like particles.

In another embodiment, the vaccine composition further comprises at least one adjuvant, such as alum, squalene-based oil-in-water nanoemulsion, MF59 adjuvant or ASO3 adjuvant.

The present invention provides a method for preventing viral infection in an individual, comprising administering a vaccine composition to the individual. The invention also provides the use of a vaccine composition as described herein for the manufacture of a medicament for use in preventing infection by a virus in an individual.

In some embodiments of the invention, the viral infection is a coronavirus infection or an influenza virus infection. In particular, the viral infection is a SARS-CoV-2 infection or an H5N2 or H3N2 influenza virus infection.

In some embodiments of the invention, VLPs are administered to prevent viral replication or alleviate symptoms caused by viral infection.

The invention also provides a method for generating antibodies, comprising administering a vaccine composition to an individual and harvesting antibodies specific for VLPs. The invention also provides the use of a vaccine composition as described herein for the manufacture of a medicament required for an individual to produce antibodies specific for a VLP.

The present invention may be more readily understood by reference to the following detailed description, examples, and chemical diagrams and tables of various embodiments of the invention and their associated descriptions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As utilized in accordance with the present invention, unless otherwise specified, the following terms shall be understood to have the following meanings:

As used herein, the use of "or" means "and/or" unless stated otherwise. In the case of multiple dependencies, the use of "or" merely serves to refer alternatively to more than one of the preceding independent or subordinate terms.

It must be noted that, as used in this specification and the accompanying patent claims, the singular forms "a", "a" and "the" include plural referents unless the context clearly dictates otherwise. Therefore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

As used herein, the term "optional/optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For example, the phrase "optionally a pharmaceutical agent" means that the pharmaceutical agent may or may not be present.

As used herein, the expression "stable expression (stably expressing)" is intended to mean genetic material that is being stably expressed and/or permanently and stably integrated into the genome of a host cell, and therefore has characteristics consistent with that of the host cell over time. The same performance potential as the native genetic material.

As used herein, the term "virion-like particle" refers to a structure similar to a virus particle. Furthermore, viral particles according to the invention are non-replicative and non-infectious since they lack all or part of the viral genome, in particular the replicative and infectious components of the viral genome. Viral particles according to the invention may contain nucleic acids that are different from their genome. Typical and preferred embodiments of such viral particles according to the invention are viral capsids, such as those of corresponding viruses, bacteriophages or RNA bacteriophages. The terms "viral capsid" or "capsid" as used interchangeably herein refer to a macromolecular assembly composed of viral protein subunits. Typically and preferably, viral protein subunits are assembled respectively into viral capsids and capsids with an intrinsic repetitively organized structure, where the structure is usually spherical or tubular. For example, the capsids of RNA bacteriophages have a spherical form with icosahedral symmetry. As used herein, the term "capsid-like structure" refers to a macromolecular assembly composed of viral protein subunits that reassembles capsid morphology in the previously defined sense but deviates from typical symmetrical assembly while maintaining a sufficient degree of order and Repeatability.

In one embodiment of the invention, the virus-like particle is a multimer of viral structural proteins, preferably a multimer of viral coat protein and/or viral envelope protein, which does not contain polynucleotides, but has in other aspects Characteristics of the virus, such as binding to cell surface receptors, being internalized with receptors, being stable in the blood, and/or containing glycoproteins.

The term "viral structural protein" is used in the context of the present invention to refer to viral coat protein or viral envelope glycoprotein.

As used herein, the term "cell line" refers to cultured cells that can be passaged (divided) more than once. The present invention relates to cell lines that can be passaged more than 2 times, up to 200 times or more, including any integer therebetween.

The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (eg, mRNA) molecules into cells, preferably into animal cells. In the context of the present invention, the term "transfection" encompasses any method known in the art for introducing nucleic acid molecules into cells, preferably into animal cells, such as into mammalian cells. Such methods include, for example, electroporation, lipofection, for example, based on cationic lipids and/or liposomes, calcium phosphate precipitation, transfection based on nanoparticles, transfection based on viruses or based on cationic polymers such as DEAE-polydextrose. or polyethylenimine and others) transfection. Preferably, introduction is non-viral.

As used herein, the term "expression cassette" refers to a recombinant DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The term "preventing/prevention" is recognized in the art, and when used with respect to a condition, it includes the administration of an agent before the onset of the condition to reduce the medical condition in an individual relative to an individual who does not receive the agent. the frequency or severity of symptoms or delaying their onset.

As used herein, the term "individual" means any animal, preferably a mammal, and more preferably a human. Examples of individuals include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cattle, horses, dogs, and cats.

As used herein, the term "immunologically effective amount" refers to an amount of a composition that, when introduced into an individual, is sufficient to induce an immune response in the individual. The amount of a composition required for immunization to be effective will vary depending on many factors, including the composition, the presence of other components in the composition (eg, adjuvants), the antigen, the route of immunization, the individual, previous immune or physiological state, and the like.

As used herein, the term "adjuvant" refers to a non-specific stimulator of an immune response or a substance that allows the creation of a reservoir in the host, which when combined with the compositions of the present invention provides even more enhanced and/or prolonged immunity. reaction, optimal interleukin production. A variety of adjuvants are known in the art and can be used in the present invention. Preferred adjuvants are selected from the group consisting of incomplete Freund's adjuvant, aluminum-containing adjuvants, modified cell wall dipeptides, surface-active substances such as lysolecithin, Pluronic polyols, polyanions, peptides , oil emulsion, keyhole hemocyanin, dinitrophenol, BCG (Bacillus Calmette-Guérin), Corynebacterium parvum, ligands of Tudor-like receptors (TLR), including but not limited to peptidoglycan, lipopolysaccharide and its derivatives, Poly I:C, immunostimulatory oligonucleotides, imidazoquinolines such as resiquimod and imiquimod, flagellin, monophospholipid immunomodulators, AdjuVax 100a, QS-21 , QS-18, GPI-0100, CRL1005, MF-59, OM-174, OM-197, OM-294, viroid adjuvant technology and any mixture thereof. For the purposes of the present invention, highly preferred adjuvants are aluminum-containing adjuvants, preferably aluminum-containing mineral gels, most preferably aluminum gels. In a highly preferred embodiment, the adjuvant is aluminum glue. The term adjuvant also encompasses mixtures of any of the substances listed above. The particles of the invention, preferably VLPs, have generally been described as adjuvants. However, the term "adjuvant" as used in the context of this application refers to an adjuvant that is not a particle of the present invention, particularly a VLP used in a composition. In each case, the term adjuvant refers to the adjuvant used in addition to the particles.

As used herein, the term "immune response" refers to any action of an individual's immune system against a molecule or compound, such as an antigen. In mammals, the immune response involves cellular activity and the production of soluble molecules such as interleukins and antibodies. Thus, the term includes humoral and/or cellular immune responses that result in activation or proliferation of B lymphocytes and/or T lymphocytes. However, in some cases the immune response may be of low intensity and only become detectable upon use of at least one substance according to the invention. "Immunogenic" means an agent used to stimulate the immune system of a living organism, thereby increasing one or more functions of the immune system and targeting the immunogenic agent.

Virus-like particles have recently emerged as promising multifunctional tools in the fight against viral infections. Recombinant VLPs produced in cell culture systems do not contain viral genomes and cannot replicate. For vaccines, VLPs are an effective and safer alternative to live attenuated viruses and fully deactivated viruses.

Therefore, the present invention aims to provide an animal cell that stably expresses virus-like particles. The present invention not only significantly improves the quality of the manufacturer's master cell bank and provides the highest speed to market and performance, but also avoids unintended consequences due to the time-consuming selection process of randomly inserting transfected DNA into the genome of the cGMP bank master cell line. shortcoming. In some embodiments of the present invention, stable single cells of animal cell lines with designed transgenic genes are preferred. These transgenic genes have the characteristics of inserting genes at predetermined identical gene sites and exhibit Maximize the amount of gene expression to generate production cell lines for the manufacture of drugs for producing antibodies and preventing viral infections in individuals.

The present invention provides a method for generating stable cell lines that co-express multiple genes, which is of great use for the production of vaccine candidates. In order to achieve a more satisfactory immune response, animal cells are used to stably express VLPs in the present invention. In order to enable high-yield and rapid generation of expressive cell lines through site-directed chromosomal gene insertion, mammalian and insect cell lines are provided to co-express multiple viral genes and efficiently produce VLPs. In some embodiments of the invention, the VLPs mimic the coronavirus SARS-CoV-2 and the avian influenza virus H5N2 or H3N2.

VLP contains four major coronavirus structural proteins, including spike (S) protein, membrane (M) protein and envelope (E) protein, which are regarded as promising vaccine candidates and in the nature of SARS-CoV-2. Useful diagnostic agent for detecting human antibodies following infection. The simultaneous expression of the S, M and E proteins of coronavirus in the same cells of higher animals can initiate the self-assembly and release of VLPs.

Viral entry of SARS-CoV-2 is mediated by the surface glycoprotein spike (S), which binds the host cell's angiotensin-converting enzyme 2 (ACE2) and undergoes a pre- to post-fusion conformational change ( Walls et al. , Cell 176, 1026-1039 e1015, 2019 ). Although the receptor-binding domain (RBD) of S is recognized as providing immunogenicity in vaccine development, the S1 domain targeting outside the RBD and the S2 domain of SARS-CoV-2 S have also been identified in the blood of convalescent patients. Other neutralizing antibodies ( Brouwer et al. , Science 369, 643-650, 2020 ; Huang et al. , PLoS Pathog 17, e1009352, 2021 ). An S protein with a 2P mutation located at the corner between the central helix of S2 and heptapeptide repeat 1 has been constructed to stabilize the S protein in its prefusion structure and avoid shedding of the S1 subunit in the COVID-19 vaccine ( Tostanoski et al. Human , Nat Med 26, 1694-1700, 2020 ; Koff et al. , Sci Transl Med 13, 2021 ; Corbett et al. , N Engl J Med 383, 1544-1555, 2020 ; Polack et al. , N Engl J Med 383, 2603 -2615, 2020 ; Voysey et al. , Lancet 397, 99-111, 2021 ).

In some embodiments of the present invention, a site-specific stable transfection system is used to provide two enveloped VLPs presenting full-length trimers of non-cleavable 2P-S and D614G-S, and are produced by suspension culture of the human 293F cell line , as immunogen candidates. The S protein of VLP 2P-S carries 5 mutations (3 that disable furin-type protease S1/S2 cleavage and 2 prolines used to stabilize the structure), and D614G-S VLP presents the native form S of the D614G variant. .

Examples of spike proteins include, but are not limited to, native D614G spike protein (SEQ ID NO: 6), diproline mutant spike protein (2P-S) (SEQ ID NO: 8), D614G-S mutant spike protein , Delta-or spike protein (SEQ ID NO: 10), Delta-GSAS 2P mutant spike protein (SEQ ID NO: 12), Omicron-or spike protein (SEQ ID NO: 14), Omicron-GSAS 2P mutation Spike protein (SEQ ID NO: 16). In one embodiment of the invention, the codons of the native D614G spike protein are optimized as shown in SEQ ID NO: 7; the codons of the diproline mutant spike protein (2P-S) are as shown in SEQ ID NO : Optimized as shown in 9; the codons of Delta-or spike protein are optimized as shown in SEQ ID NO: 11; the codons of Delta-GSAS 2P spike protein are shown in SEQ ID NO: 13 Optimized; the codons of Omicron-or spike protein are optimized as shown in SEQ ID NO: 15; the codons of Omicron-GSAS 2P spike protein are optimized as shown in SEQ ID NO: 17.

In some embodiments of the invention, the VLPs comprise hemagglutinin (HA), neuraminidase (NA) and matrix proteins (M1 and M2) of influenza virus. Co-expression of structural proteins in the same animal cell results in the self-assembly and release of VLPs, exhibits the native structure of the entire virion and has been shown to be useful in eliciting neutralizing antibodies, thus representing a promising vaccine candidate and recombination for the development of diagnostic reagents antigen.

In some embodiments of the invention, the influenza virus is H5N2, such as A/duck/Taiwan/01006/2015/H5N2. Influenza virus structural proteins as disclosed herein include hemagglutinin (HA), neuraminidase (NA) and matrix proteins (M1 and M2). In some embodiments of the invention, the H5 protein is as shown in SEQ ID NO: 18, and the codons of the H5 protein are optimized as shown in SEQ ID NO: 19. In some embodiments of the invention, the N2 protein is as shown in SEQ ID NO: 20, and the codons of the N2 protein are optimized as shown in SEQ ID NO: 21.

In one embodiment of the invention, the influenza virus is H5N2, such as A/Taiwan/083/2006/H3N2. In some embodiments of the invention, the M1 protein is as shown in SEQ ID NO: 22, and the codons of the M1 protein are optimized as shown in SEQ ID NO: 23. In some embodiments of the invention, the M2 protein is as shown in SEQ ID NO: 24, and the codons of the M1 protein are optimized as shown in SEQ ID NO: 25.

In some embodiments of the present invention, animal cell lines are produced by a "rapid multigene overexpression system" that includes (1) capturing the highest expression capacity site and inducible expression of the GFP gene in the chromosome of the host cell line chromosomal gene loci to obtain founder cell lines, (2) engineering donor plastids that carry multiple transgene clusters driven individually by the CMV/TO promoter, and (3) mediated by FLPe recombinase Guided cassette exchange (RMCE) "exchanges the captured GFP gene" with the donor gene cluster to achieve targeted insertion of all transgenic genes.

Specifically, animal cell lines as described herein are produced by (1) capturing chromosomal loci that allow the highest levels of inducible foreign gene expression in mammalian and insect cell lines, and (2) engineering to carry FRT is flanked by several transgene cassette clusters, CMV/TO promoter-driven donor plastids expressing 3 or 4 viral structural proteins, (3) "gene exchange" donors are co-transfected with FLPe recombinase cassette to achieve site-specific insertion of all transgenic genes, and (4) production and purification of VLPs.

In one embodiment of the invention, the inducible expression cassette includes as a target cassette a tetracycline-inducible promoter, such as the CMV/TO cassette of the Flp/FRT recombination system, adjacent upstream and downstream of F- and F3 sequences. In some embodiments of the invention, animal cells also stably express the tetracycline inhibitor cassette. In some embodiments of the invention, a stably expressing tetracycline suppressor cassette comprises a tetracycline suppressor gene and a self-cleaving 2A peptide-linked pumimycin S resistance gene derived from porcine Tiescavirus 1. In some embodiments of the invention, the stable performance cassette is an EF1a/eIF4g-pCI-TetR-P2A-BSR cassette.

In one embodiment of the invention, a targeting cassette for a mammalian cell system is shown in Figure 13, and a map of plastid pGEMT-RMCE1-CMVto-sfGFP is shown in Figure 15.

In one embodiment of the invention, the inducible expression cassette comprises a doxycycline-inducible promoter. Examples of doxycycline-inducible promoters include, but are not limited to, the promoters of the OpMNPV immediate early gene 2 (IE2) and the Cecropia gigas actin A1 gene. In principle, the tet operator (TetO2), which expresses the Tet repressor and is inserted in tandem with 2 repeats downstream of the TATA box, is linked to control the interaction between IE2 and the Cecropia actin A1 promoter by allowing the Tet repressor to bind. expression until doxycycline treatment releases the Tet repressor and turns on gene expression. The inducible expression cassette further contains CMV/TO driven VLP genes distributed in 2 or 3 tandem genes. The entire transgenic gene cluster is adjacent to two FRT sites (F and Fn) upstream and downstream to become the target cassette of FLPe recombinase. The donor plasmid and FLPe expression plasmid were co-transfected into the founder cell line. Cells were sorted again based on reporter deletion and HA gain expression in the VLP gene.

In one embodiment of the present invention, the targeting cassette of the insect cell system is shown in Figure 14, and the map of plastid pUC57.Insect RMCR1 is shown in Figure 16.

The invention also provides a method for producing virus-like particles, which includes culturing animal cells and harvesting virus-like particles.

In some embodiments of the invention, the method includes culturing animal cells, removing cell debris and other large aggregates, and ultracentrifuging through a two-step sucrose gradient (30% and then 40%-60%) or using Crapto Q and Capto Core 700 multi-modal (MMC) and diafiltration 2-step column chromatography, or use Capto DeVirS and Capto Core 700 2-step column chromatography to purify VLPs.

The invention also provides a virus-like particle produced by a method comprising culturing animal cells as described herein and harvesting the virus-like particle.

The present invention provides a vaccine composition comprising an immunologically effective amount of virus-like particles produced by a method comprising culturing animal cells as described herein and harvesting virus-like particles.

In another embodiment, the vaccine composition further comprises at least one adjuvant, such as alum or incomplete Freund's adjuvant.

The present invention provides a method for preventing viral infection in an individual, comprising administering a vaccine composition to the individual. The invention also provides the use of a vaccine composition as described herein for the manufacture of a medicament for preventing viral infection in an individual.

In some embodiments of the invention, the viral infection is a coronavirus infection or an influenza virus infection. In particular, the viral infection is a SARS-CoV-2 infection or an H5N2 or H3N2 influenza virus infection.

In some embodiments of the invention, VLPs are administered to prevent viral replication or alleviate symptoms caused by viral infection. An example of symptoms due to SARS-CoV-2 infection includes, but is not limited to, pneumonia.

The invention also provides a method for generating antibodies comprising administering a vaccine composition to an individual and harvesting antibodies specific for VLPs. The invention also provides the use of a vaccine composition as described herein for the manufacture of a medicament for an individual to produce antibodies specific for VLPs.

In particular, the antibodies are neutralizing antibodies. In addition, neutralizing antibodies can be used to treat viral infections and symptoms caused by viral infections.

The following examples are provided to assist those skilled in the art in practicing the invention. Example materials and methods:

Induced Performance Cassette

To construct the inducible expression cassette, we adapted the tetracycline-inducible promoter (CMV/TO) obtained from pcDNA4/TO (Invitrogen) into a universal chimeric gene obtained from pCI (Promega, Madison, WI). Intron, recombinant open reading frame and polyadenylation signal (BGH polyA) or SV40 polyA from the bovine growth hormone gene are linked. To stop the CMV/TO promoter prior to tetracycline or doxycycline induction, we introduced constitutive expression of the Tet repressor (TetR, derived from pcDNA6/TR and optimized for mammalian codon usage). To ensure that TetR is expressed in stably transfected cells, TetR was designed to co-repress the bomimycin S resistance gene (bsr, Borrelia coriaceae cytidine deaminase), which is derived from Self-cleaving 2A peptide (P2A) ligation of porcine Tiscavirus 1 was used for negative selection. Using CMV/TO-GFP as a reporter that binds to the EF1a/IF4g-pCI-TR-P2A-BSR gene (pUC57.Insect RMCR1 or pGEMT-RMCE1-CMVto-sfGFP), we have randomly inserted into the host cell genome Founder cells are generated and those showing maximal expression of Tet-inducible GFP are selected. During our optimization of green fluorescence in different host cell lines, superfolder GFP was used for mammalian cell lines and turbo GFP was used for insect cell lines. Both GFP and BSR used were optimized for mammalian codon usage.

The sequence of Tet suppressor is shown in SEQ ID NO: 26, and the optimized codon is SEQ ID NO: 30; the sequence of BSR is shown in SEQ ID NO: 27, and the optimized codon is SEQ ID NO: 31; The sequence of sfGFP is shown in SEQ ID NO: 28, and the optimized codon is SEQ ID NO: 32; the sequence of P2A is shown in SEQ ID NO: 29, and the optimized codon is SEQ ID NO: 35; hEF1α/ The sequence of the eIF4γ promoter is shown in SEQ ID NO: 33; the sequence of CVM/TO is shown in SEQ ID NO: 34; the polyadenylation signal sequence of SV40 is shown in SEQ ID NO: 36; bovine growth hormone (BGH) ) is shown in SEQ ID NO: 37;

The sequence of the M1 protein of A/California/07/2009/H1N1 is shown in SEQ ID NO: 40; the sequence of turboGFP is shown in SEQ ID NO: 41; the sequence of the constitutive Cecropia moth promoter is shown in SEQ ID NO: 42; the sequence of the inducible Cecropia promoter is shown in SEQ ID NO: 43; the sequence of inducible IE2 is shown in SEQ ID NO: 44; the polyadenylation signal sequence of OpMNPV is shown in SEQ ID NO: 45.

tetracycline inhibitor cassette

Constitutive expression cassettes are not used in viral genes. Indeed, it is used to express the tet repressor to stop the CMV/TO promoter until induction by tetracycline or doxycycline.

Establishment of expressive cells

VLP expressing cell lines were established in FreeStyle 293F cells. The 293F founder cell line serves as the target cassette of the Flp/ FRT recombination system through stable transfection of CMV/TO-GFP, flanked by F- and _F3 sites, and EF1a/eIF4g-TetR-P2A-BSD. Constitutive performance is established. Double-transfected cells were negatively selected by puminomycin S, and single cells were separated by GFP intensity. For site-directed recombination of S/M/E genes, founder cells were expressed using donor plastids carrying the CMV/TO-S-CMV-TO-M-IRES-E expression cassette flanked by F- and _F3 sites and expressing FLPe The plasmids of the recombinase were co-transfected, and individual cells lacking GFP and obtaining the S/M/E gene were subsequently isolated.

Immunofluorescence staining

VLP expressing cells were induced with Dox or vehicle control for 48 hours. Cells were then fixed in 4% paraformaldehyde for 10 min and soaked in 0.05% Triton X-100 for 1 min. Cells were then blocked with 3% BSA, incubated with specific primary antibodies, washed, and then incubated with goat anti-rabbit or goat anti-mouse IgG conjugated to Cy2 or Cy3 dyes. Fluorescence images were captured with an inverted fluorescence microscope (Observer D1, Zeiss). The antibodies used in this study were S1 (40150-R007, Sino Biological) and S2 (GTX632604, Genetex).

cell suspension culture and VLP produce

293F VLP production cells (approximately 2 × 10 ⁶ /mL) were inoculated in FreeStyle 293 Expression Medium (Gibco) in 2 L Erlenmeyer flasks and incubated in a humidified incubator with ₅ % CO at 37°C. Suspension culture was performed by stirring at 150 rpm. To induce cell expression and secretion of VLPs, 1 µg/ml doxycycline was added to the culture medium for 72 hours, and the conditioned medium was harvested, filtered through 0.45 µm Stericap, concentrated by Vivaflow 50 (Sartorius Stedim Biotech), and used on an ÄKTA Chromatographic purification was performed on Capto Q and Capto Core 700 columns (GE Healthcare, Swenson) on the pure 25 system (Cytiva, GE Healthcare, Swenson) at 4°C.

Protein analysis and Western blotting

The protein components of purified VLPs were quantified by Quant-iT protein analysis kit (Invitrogen). Purified VLPs were mixed with Lämmle SDS-PAGE sample buffer without reducing agent and boiled (N), without reducing agent and boiled for 2 minutes (NB), with reducing agent and boiled (RB), followed by a 4-12% gradient. SDS-PAGE was performed on the gel followed by Western blot analysis.

Particle size dynamic light scattering (DLS) Determination

VLP samples were diluted to 0.1 µg/mL in 20 mM phosphate buffer pH 7.4, passed through a 0.45 µm filter, and analyzed on a Nano ZS particle size analyzer (Malvern Zetasizer, Malvern Instruments). Each sample was measured by DLS for 60 seconds, twice consecutively. Use the accompanying software (Nanov510) to convert intensity-based measurements into a size distribution that integrates the number of particles in each size class and present it in the form of a curve graph showing the frequency distribution of the sample, where the area under the curve is related to the detection within the specified size range. Proportional to the number of particles measured. The mean diameter of the VLPs was then calculated as the mean size ± standard deviation (SD) of the particle population from three independent experiments.

cryo-electron microscopy

To prepare cryo-EM grids, aliquots (approximately 4 μL) of purified VLPs were added to glow discharge Quantifoil R2/2 porous carbon grids (Quatifoil GmbH, Germany). The grids were blotted dry with filter paper on both sides for 3 seconds and subsequently snap-frozen into liquid ethane cooled by liquid nitrogen using a Thermo Scientific Vitrobot system (Mark IV). Cryo-EM grids were stored in liquid nitrogen until imaging, and all subsequent steps were performed below -160°C to prevent devitrification.

Cryo-EM grids were cut, mounted in cassettes, transferred with nanocaps and loaded into a 200 kV Talos Arctica transmission electron microscope (Thermo Fisher Scientific). The alignment was performed in nanoprobe mode (spot size 3, gun lens 4, C2 lens set to 43.8%) to achieve parallel beam alignment and coma-free alignment. Cryo-EM images were recorded at 92,000× magnification with a Falcon III detector (Thermo Fisher Scientific) operating in linear mode with a pixel size of 1.1 Å/pixel. The defocus setting for imaging was approximately 2.5 μm and the dose rate was set to approximately 20 e-/Å2 per second to produce a total dose of approximately 50 e-/Å2 in 2.5 seconds. Cryo-electron microscopy images were collected using EPU-2.2.0 software (Thermo Fisher Scientific).

Cryo-EM grids were loaded into a 200 kV Talos Arctica transmission electron microscope (Thermo Fisher Scientific). Oblique images were recorded using a Falcon III detector (Thermo Fisher Scientific) at a magnification of 73,000× with a pixel size of 1.4 Å/pixel. The defocus setting used for imaging was approximately 6 μm. Tomography-4.10.0 software (Thermo Fisher Scientific) was used to collect slices with a tilt range of ±60° and constant angle increments of 3° (from +20° to -60°, and subsequently from +20° to +60°). Scan oblique images. Each tilt uses a dose of approximately 3 e-/Å2, resulting in a total dose of 41 tilt images of approximately 120 e-/Å2. 3D tomographic images were reconstructed from oblique images using Inspect3D-4.2 software (Thermo Fisher Scientific). Align the oblique image, adjust the oblique axis, and perform tomographic reconstruction using Simultaneous Iterative Reconstruction Technology (SIRT). The tomographic reconstruction was visualized using the “Inspect Stack” function in Inspect3D-4.2 software (Thermo Fisher Scientific).

Immunization strategy

Eight-week-old female C57BL/6 mice and male golden Syrian hamster lines were purchased from the National Laboratory Animal Center, Academia Sinica, Taiwan. VLP formulations were adjuvanted with or without Alhydrogel or AddaVax adjuvant (InvivoGen) as a 1:1 mixture. Nine-week-old mice and hamsters were immunized twice by subcutaneous injection, and the interval between the initial injection and the additional injection was 10 days ( Liang et al ., Sci Transl Med 9, 2017 ). The injection volume for mice was 100 μL and for hamsters was 200 μL. Blood samples were collected before the booster injection and weekly after the booster injection.

ELISA Determination of specificity

ELISA plates (Nunc) were coated with D614G-S VLP overnight at 4°C and blocked with StartingBlock blocking buffer (Thermo Fisher Scientific). Hamster serum samples at indicated dilutions were added to coated ELISA plates and subsequently incubated at 37°C for 1 hour, chased with HRP-conjugated secondary Ab and developed with TMB substrate (Pierce), and finally at 450 nm. Measure the absorbance (Power Wave XS, Bio-Tek). Wash three times with PBST buffer between each ELISA step. The IgG subtypes in hamster serum were detected using separate secondary antibodies, namely anti-hamster IgG1 (1940-05, SouthernBiotech) and anti-hamster IgG2 and IgG3 (1935-05, SouthernBiotech).

ELISpot analyze

Mouse IFN-γ or IL-4 values were determined using an ELISpot assay kit (R&D Systems). The immunized mice were euthanized by carbon dioxide the second week after the booster dose. Splenocytes were harvested and seeded at 5 × 10 ⁵ /well in 96-well plates, precoated with anti-IFN-γ or anti-IL-4 Ab for 24 hours. After washing three times with buffer and incubating with detection antibody overnight at 2-8°C, the plate was washed three times with buffer and incubated with avidin-AP for 1 hour at room temperature. It was then washed three more times and color was then developed by incubation with BCIP/NBT chromogen for 1 hour at room temperature and the plates were rinsed with deionized water.

Pseudovirus neutralization analysis

293T/17-ACE2 cells and pseudovirus were provided by the National RNAi Core Facility (Academia Sinica, Taiwan). 293T/17-ACE2 cells were seeded in 96-well plates with 1% heat-inactivated FBS DMEM medium. Immune serum was diluted 2 times from 1:20. An equal volume of pseudovirus was incubated with diluted immune serum at 37°C for 1 hour. The pseudovirus carries the S protein of SARS-CoV-2 and the luciferase gene as a reporter. After incubation, the serum-pseudovirus mixture was added to the cells and centrifuged at 1,100 × g for 30 minutes, and the cells were cultured at 37°C in a humidified incubator with 5% _CO for 24 hours. The medium was refreshed with 1% FBS DMEM medium and incubated for an additional 48 hours. The luciferase activity was measured using a firefly luciferase assay kit (Biotium).

CPE Neutralization analysis

Serially diluted antibodies were incubated with 100 TCID ₅₀ SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020) for 1 hour at 37°C. The mixture was then added to preseeded Vero E6 cells and cultured for 4 days. Cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. Dishes were washed with tap water and infections were scored. The 50% protective power price was calculated using the Reed and Muench method.

Virus attack experiment

The viral challenge infection experiment in the hamster model was approved by the Institutional Animal Care and Use Committee (IACUC) and the P-3 Laboratory of Academia Sinica. Vaccinated Syrian hamsters were anesthetized and treated with 1 × 10 ⁵ PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID registration number: EPI_ISL_411927) (lot number: IBMS20200819, 8.0 × 10 ⁵ PFU/mL in a volume of 125 μL) for intranasal challenge. All hamsters were weighed daily after SARS-CoV-2 challenge infection. Hamsters that survived the infection experiments were euthanized using carbon dioxide.

Quantifying virus titer in tissues by cell culture infection assay

Homogenize hamster tissue in 600 μL of DMEM with 2% FBS and 1% penicillin/streptomycin using a homogenizer. The tissue homogenate was centrifuged at 15,000 rpm for 5 minutes and the supernatant was collected for viable virus titration. Briefly, 10-fold serial dilutions of each sample were added to Vero E6 cell monolayers in quadruplicate and grown for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. Dishes were washed with tap water and infections were scored. The 50% tissue culture infectious dose (TCID ₅₀ )/mL was calculated by the Li-Ming method.

used for SARS-CoV-2 RNA Quantitative real-time RT-PCR

To measure the RNA content of SARS-CoV-2, a package targeting the SARS-CoV-2 genome was used by the TaqMan real-time RT-PCR method described in a previous study ( Corman et al ., Euro Surveill 25, 2020 ). Specific primer for the region 26,141 to 26,253 of the membrane (E) gene. Use forward primer E-Sarbeco-F1 (5'-ACAGGTAC GTTAATAGTTA ATAGCGT-3', SEQ ID NO. 1) and reverse primer E-Sarbeco-R2 (5'-ATATTGCAGCAGTACGCACACA-3', SEQ ID NO. 2) , plus probe E-Sarbeco-P1 (5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO. 3)-BBQ-3'). A total of 30 μL RNA solution was collected from each sample using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. A 5 μL aliquot of RNA was added to a total of 25 μL mixture of the Superscript III one-step RT-PCR system and Platinum Taq polymerase (Thermo Fisher Scientific). The final reaction mixture contained 400 nM forward and reverse primers, 200 nM probe, 1.6 mM deoxynucleoside triphosphates (dNTPs), 4 mM magnesium sulfate, 50 nM ROX reference dye, and 1 μL enzyme mix. Cycling conditions were performed using a one-step PCR protocol: 55°C for 10 min to synthesize first-strand cDNA, followed by 3 min at 94°C, and 45 cycles of amplification at 94°C for 15 sec and at 58°C for 30 sec. Data were collected and calculated by an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific). The synthesized 113 bp oligonucleotide fragment was used as a qPCR standard to calibrate the number of viral genome copies. Oligonucleotides were synthesized by Genomics BioSci and Tech (Taipei, Taiwan).

Histopathological analysis and IHC

Tissues were fixed in 4% paraformaldehyde for 3 days, transferred to PBS, embedded in paraffin within 7 days, and sectioned into blocks at 4 μm. Slides were baked at 37°C overnight, then deparaffinized in xylene and rehydrated through a graded series of ethanol to distilled water. Histopathological analysis was performed by hematoxylin and eosin staining (Muto Pure Chemicals) and subsequent blue staining using Tacha blue solution (Biocare). For SARS-CoV-2-N IHC, antigen retrieval was performed in Reveal Decloaker buffer (Biocare) using an autoclave at 90°C for 15 min. For CD3, MX1, IBA1, and MPO IHC, antigen retrieval was performed in target retrieval solution pH 9.0 (Dako) using an autoclave at 85°C for 10 min. Slides were washed with PBS and subsequently treated with 3% hydrogen peroxide for 10 minutes. Slides were washed with PBS and blocked with background destroyer (Innovex) for 1 hour. Primary antibodies anti-SARS-CoV2 N (40588-T62, Sino Biological, 1:500), anti-CD3 (ab16669, Abcam, 1:50), anti-MX1 (13750-1-AP, Proteintech, 1:100), anti- IBA1 antibody (10904-1-AP, Proteintech, 1:500) or anti-MPO (ab208670, Abcam, 1:500) was incubated overnight at 4°C. Multivalent secondary antibodies (Innovex) were applied for 15 minutes and treated with peroxidase (Innovex) for 15 minutes. The Betazoid DAB chromogen set was used and subsequently counterstained with hematoxylin, followed by bluing using Tacha bluing solution (Biocare).

Statistical analysis

Analyzes were performed using GraphPad Prism 7.05 (GraphPad Software). Two-way analysis of variance (ANOVA) was used to compare ELISpot, body weight, viral RNA, TCID ₅₀ and substantive inter-group data. CPE and pseudovirus neutralizing potency were assessed by one-way ANOVA. Neutralization data were log ₂ transformed. Viral RNA and TCID ₅₀ were log ₁₀ transformed. P values less than 0.05 were considered significant.

Example 1 Produced in mammalian cells COVID-19 VLP

In order to efficiently produce VLP antigens on a large scale, 293F stable monomers were established by inserting gene clusters at specific sites in the genome to co-express the spike (S), membrane (M) and envelope (E) proteins of SARS-CoV-2. strain cells. The S protein sequences encoding pre-fusion stable 2P-S (SEQ ID NO: 8) and native D614G variant S (SEQ ID NO: 6) were constructed as antigen candidates for VLP vaccines (Figure 1A). Using our platform, VLPs from 293F stable monoline cells achieved high yields of 25 to 31 mg/L. The expression of S protein in producer cells was detected by immunofluorescence analysis (IFA) using anti-S1 and anti-S2 antibodies (Figure 1B). Furthermore, after purification from the conditioned medium, the S protein of the VLPs was analyzed by Western blotting using specific antibodies (Figure 1C and Figure 1D). According to the design, the S protein is full-length S (approximately 180K) in 2P-S or the protein is cleaved into S1 (100K) and S2 (85K) in D614G-S. Some oligomers are detected when the molecular weight is higher than 250K. Both VLPs (2P-S and D614G-S) are recognized by commercially available anti-S1 and anti-S2 as well as human antibodies from the plasma of convalescent COVID-19 patients. It is worth noting that denaturation of S protein by treating VLPs with reducing agents and boiling destroys the S protein structure and reduces antibody recognition. Anti-S1 is more significant than anti-S2 (Figure 1C and Figure 1D, and Figure 6A and Figure 6B ). By summing the S protein signals (detected by S1 and S2 monoclonal antibodies respectively) and interpolating from the standard curve of the recombinant S-2P protein, the S protein content in each VLP was quantified to be approximately 20% of the total VLP protein ( The average was 19.3% in 2P-S and 18.0% in D614G-S) (Figure 7A and Figure 7B). Using dynamic light scattering (DLS) analysis, the average particle sizes of 2P-S and D614G-S were 127.2 and 123.9 nm, respectively (Figure 1E). By cryo-electron microscopy (cryo-EM) analysis, the morphology of 2P-S and D614G-S was characterized by a clear spike crown (Figure 1F). Although the D614G-S VLP exhibits flexible spikes, which may be attributed to the conformational flexibility of the three recently identified hinges (hip, knee, and ankle) tilted at different angles within the stalk (S2) domain, the 2P- S presents uniform upright spikes ( Turonova et al. , Science 370, 203-208, 2020 ). These results indicate that 2P-S VLP and D614G-S VLP respectively present full-length pre-fusion forms with upright protrusions of spines and bends in varying degrees and directions.

Example 2 VLP induced in mouse models Th1 and Th2 reaction

Inducing a balanced Th1/Th2 response through vaccination is a necessary criterion for the development of a COVID-19 vaccine to avoid the possibility of VAERD induced by a Th2-biased response after vaccination ( Acosta et al ., Clin Vaccine Immunol 23, 189-195, 2015 ; Bottazzi et al. , Microbes Infect 22, 403-404, 2020 ). In order to evaluate the potential of these two VLPs and identify favorable adjuvants for the VLP formulation to enhance the immune response, a homologous prime-plus vaccination strategy was performed to determine the immunogenicity in the C57BL/6 mouse model (Figure 2A). We immunized mice with D614G-S VLPs containing only 0.75 μg (low) or 2.25 μg (high) S protein and VLPs containing 0.75 μg S protein supplemented with aluminum hydroxide gel (alum) or AddaVax. We used D614G-S VLP as the diagnostic antigen due to its similarity to the ancestral wild-type SARS-CoV-2. In ELISA analysis, VLPs adjuvanted with AddaVax induced high antibody titers of approximately 2 × 10 ⁵ similar to the alum group, which was higher than VLPs without adjuvant (5 × at high and low doses of antigen, respectively). 10 ⁴ and 1 × 10 ⁵ ) (Table 1). A similar effect was seen in Western blot analysis using pooled antisera from the same group, with stronger signals seen in the AddaVax and Alum groups (Figure 2B). Additionally, cell-mediated immune responses were analyzed by ELISpot analysis. Interestingly, D614G-S VLP alone elicited both Th1 and Th2 responses, and only AddaVax further enhanced the IFNγ (Th1) response. Although both alum and AddaVax enhanced IL-4 (Th2) response, AddaVax had a greater adjuvant effect on IL-4 than alum (Figure 2C). These findings indicate that VLPs elicit Th1 and Th2 responses and that AddaVax is a more effective adjuvant than alum for formulating VLP vaccines. Table 1. ELISA price after vaccination Species group Total IgG IgG1 IgG2+IgG3 C57BL/6 D614G-S-Low 5 × 10 ⁴ D614G-S-High 1 × 10 ⁵ D614G-S+alum 2 × 10 ⁵ 2P-S+alum 2 × 10 ⁵ D614G-S+AddaVax 2 × 10 ⁵ 2P-S+AddaVax 2 × 10 ⁵ hamster D614G-S+AddaVax 2.4 × 10 ⁵ 2 × 10 ⁵ 5 × 10 ⁴ 2P-S+AddaVax 2.4 × 10 ⁵ 2 × 10 ⁵ 7.5 × 10 ⁴

Example 3 use AddaVax as Adjuvants VLP Induction of neutralizing antibodies in hamster model

To determine the protective efficiency of the VLP-based vaccine, we subsequently immunized golden Syrian hamsters with AddaVax-adjuvanted VLP at a dose equivalent to 1.5 μg of S protein. The immunization and challenge scheme is shown in Figure 3A. Two weeks after primary-additional immunization, both vaccines elicited high IgG titers in serum, approximately 2.4 × 10 ⁵ -fold in ELISA analysis (Table 1). Further diagnosis of the IgG subclasses induced by the VLP vaccine showed that compared with the IgG2+IgG3 (Th1 response) valence of 7.5 × 10 ⁴ and 5 × 10 ⁴ in the 2P-S and D614G-S groups, respectively Antisera from both vaccine groups contained higher IgG1 (Th2 response) titers (2 × 10 ⁵ ) (Table 1). Antisera drawn 2 weeks after booster vaccination were initially analyzed for NAb potency against the cytopathic effect (CPE) observed in Vero E6 cells infected with SARS-CoV-2. The NAb titer of the 2P-S group was approximately 468, which was 4 times higher than that of the D614G-S group. Among the 10 hamsters in the D614G-S group, 4 had NAb titers lower than 80 (Figure 3B). Through S-pseudovirus neutralization analysis, the correlation between the longitudinal NAb titer and the binding of viral S protein to the host hACE2 receptor was further analyzed. At 2 weeks after the booster dose, the NAb potency of 2P-S required to inhibit 90% of S-pseudovirus infection was higher than that of the D614G-S and simulation groups, and at least 6 weeks after the booster dose, the NAb potency remain high (Figure 3C). In a parallel examination using Western blot analysis, the antisera of the 2P-S group primarily recognized the S1 subunit within the D614G-S VLP, which mimics the viral envelope. In contrast, the D614G-S antiserum recognized S1 and S2 one week after the booster dose, but the main recognition signal shifted from S1 to S2 three weeks after the booster dose (Fig. 3D). These results indicate that 2P-S adjuvanted with AddaVax is able to elicit higher potency NAb and stable recognition of S1 compared to the D614G-S vaccine, which may alter the epitope target of the antibody.

Example 4 use AddaVax as an adjuvant VLP Prevents post-challenge viral replication in a hamster model and COVID-19 Symptoms

To assess the preventive efficacy of the VLP vaccine, hamsters immunized with PBS (mock), 2P-S and D614G-S adjuvanted with AddaVax were intranasally inoculated with 1 × 10 ⁵ plaque-forming units 46 days after booster vaccination. (PFU) of SARS-CoV-2, and autopsies were performed at 3 days post-infection (3 dpi) and 6 dpi (Fig. 4A). Viral infection resulted in significant weight loss in all mock (PBS) vaccinated hamsters. In contrast, most of the hamsters vaccinated with 2P-S and half of the hamsters vaccinated with D614G-S began to gain weight 2 days after infection (Figure 4A to Figure 4D). Viral replication, inflammation, and pathology were diagnosed in lungs and duodenum at 3 and 6 dpi based on SARS-CoV-2 viral RNA (vRNA, E gene) and replication-competent virus (tissue culture infectious dose, TCID ₅₀ ) . Both vaccines reduced vRNA in the lungs at 3 dpi. Although 2P-S appeared to be more effective than D614G-S in inhibiting lung vRNA synthesis at 3 dpi, the difference did not reach statistical significance due to the large changes in D614G-S (Fig. 4E). At 6 dpi, the vRNA content in the 2P-S lung was only 0.01% of the simulated content and close to the detection limit, while the vRNA content in the D614G-S lung was 0.1% of the simulated content (Figure 4E). vRNA was low in the duodenum, and the efficacy of the two vaccines was similar. Compared with the high TCID ₅₀ in simulated lungs (2.3 × 10 ⁶ at 3 dpi and 7.5 × 10 ³ at 6 dpi), both 2P-S and D614G-S significantly suppressed lung TCID ₅₀ at different levels at 3 dpi. content, and almost all were detected below or close to 100-fold (minimum dilution) at 6 dpi, while the duodenal viral titers in all groups were too low to detect any decrease in TCID ₅₀ after vaccination (Figure 4F). These results indicate that both 2P-S and D614G-S used as adjuvants by AddaVax can effectively reduce viral replication and infection of SARS-CoV-2.

Example 5 VLP Vaccine protects hamsters free from COVID-19 pneumonia

To further investigate the safety and efficacy of the VLP vaccine, we carefully examined the histopathology and lung tissue of vaccinated infected and uninfected hamsters at 3 and 6 dpi using H&E staining and immunohistochemistry (IHC). immune response. We observed no solidification, inflammation, or abnormalities in the lungs of uninfected hamsters, indicating that both VLP vaccines are safe (Figure 8A and Figure 8B). After SARS-CoV-2 infection, histopathological lung parenchyma in the PBS group was initially mild at 3 dpi and progressed to severe at 6 dpi, whereas parenchyma was greatly attenuated in the vaccinated group (Fig. 5A and Figure 5B). In D614G-S lungs, parenchyma was mild at 3 dpi and some progressed to moderate diffuse disease with interstitial pneumonitis at 6 dpi; on the other hand, all but one (n=5) at 6 dpi In addition to exhibiting moderate parenchymalization, all 2P-S lungs showed little or no evidence of viral interstitial pneumonia throughout 6 dpi (Fig. 5B). In situ viral replication was indicated by IHC of SARS-CoV-N, dark and diffuse SARS-CoV-N in alveolar edema around the airways that appeared in PBS lungs at 3 dpi and resolved throughout 6 dpi but remained Visible. In contrast, SARS-CoV-N was restricted to isolated cells in 2P-S lungs and small clusters in D614G-S lungs at 3 dpi, and was completely clear in both vaccinated groups at 6 dpi (Figure 5C). Type I and III interferons are general first-line defenses in response to viral infection and may inhibit SARS-CoV-2 infection and replication ( Vanderheiden et al ., J Virol 94, 2020 ). In order to reflect the interferon response, innate and acquired immunity in the host lung, we then performed IHC staining and analysis of lung samples from infected hamsters to examine interferon-induced MX1 expression and inflammatory cells, MPO ⁺ neutrophils, Infiltration of IBA1 ⁺ macrophages/monocytes and CD3 ⁺ T lymphocytes. In PBS lungs, MX1 was expressed in some alveolar epithelial cells in the infected area at 3 dpi and extensively in lower amounts in the thickened alveolar septa at 6 dpi, whereas MX1 expression at 3 dpi was restricted to 2P-S and The lower volume and density of the infected area (peribronchial) in D614G-S lungs was reduced in 2P-S lungs at 6 dpi, but could still be observed in some proliferative pneumocytes in D614G-S lungs (Fig. 5D). Small clusters of MPO ⁺ neutrophils (another professional phagocyte) were also detected in PBS and D614G-S lungs at 3 dpi, and more were observed in the inflamed areas of D614G-S lungs at 6 dpi. Multiple clusters. However, no clusters of macrophages and neutrophils were detected in 2P-S lungs at 6 dpi (Fig. 5E). At 3 dpi, some macrophage/monocyte clusters were found to infiltrate and disperse in the interstitium surrounding the airways in both vaccinated groups; however, in PBS lungs and to a lesser extent in D614G-S In the lungs, macrophage infiltration accumulated at 6 dpi and clustered in pneumonia lesions, indicating phagocytic activity (Fig. 5F). In PBS lungs, CD3+ T lymphocyte infiltration was dispersed at 3 dpi. As pneumonia progresses, T cell infiltration accumulates at 6 dpi and is dispersed in the thickened alveolar interstitium. In individuals vaccinated with 2P-S, unlike the PBS group, CD3 ⁺ T cells were mainly locally dispersed in the lung airway epithelium and remained unchanged throughout 6 dpi. However, in individuals vaccinated with D614G-S, some CD3 ⁺ T lymphocytes diffused in the lung interstitium around the airways at 3 dpi, further accumulated, and diffused extensively in the alveoli at 6 dpi, suggesting induction by viral infection. The activation, expansion and infiltration of T cells (Figure 5G). These results indicate that 2P-S and D614G-S vaccines prevent viral infection, spread and pneumonia through different mechanisms. The 2P-S vaccine protects hamsters from disease by blocking the binding of the virus to host receptors via NAb, thereby effectively preventing early viral replication and subsequent inflammation, while the D614G-S vaccine may also be involved in the use of T lymphocyte responses and activation by giant Antibody-dependent phagocytosis mediated by phagocytes/monocytes and neutrophils eliminates viruses and infected cells.

Example 6 Derived from SARS-CoV-2 Of Delta and Omicron Variant of VLP of production.

Two series of VLPs were further engineered based on the spike protein sequences of the Delta and Omicron variants of SARS-CoV-2. Production cell lines Individual cell lines were established and isolated as previously described. Co-expression of S proteins carrying tagged different mutations, including original Delta, GSAS-2P and GSAS mutants, together with M and E proteins, resulted in three different Delta-VLPs. Figures 9A to 9C show the characterization of the S protein by immunoblotting using anti-S1 and anti-S2 antibodies, and the characterization of the spherical morphology of respective VLPs by cryo-electron microscopy. By dynamic light scattering (DLS) analysis, the average diameters of Delta VLPs of original, GSAS-2P and GSAS mutants were 87.9 ± 0.6 nm, 96.5 ± 0.8 nm and 114.7 ± 0.4 nm, respectively. Similarly, we produced Omicron VLPs by co-expressing Omicron S and M and E proteins carrying different mutations of the markers (Figure 9A-9D). The average diameters of Omicron VLPs derived from S, M, and E proteins were 114.8 ± 0.8 nm, 109.3 ± 0.4 nm, and 110.7 ± 0.7 for the original, 2P-S, RQSR-2P-S, and GSAS-2P-S mutants, respectively. nm and 96.4 ± 0.4 nm.

Example 7 S Overexpression of protein is enough to drive VLP Released from animal cells.

Our proteome analysis of VLPs derived from overexpressing ancestral S, M and E proteins in the 293F cell line showed that the levels of M and E proteins were very low and almost undetectable. Therefore, our study only showed whether the S protein can drive VLP budding from transgenic cells. Interestingly, only VLPs overexpressing S protein in the 293F cell line also produce VLPs and are released in the culture medium, including ancestral S (wild type) (SEQ ID NO: 10) and its GSGS-2P-S mutant (SEQ ID NO : 12), Omicron S (original) (SEQ ID NO: 14), and Omicron 2P-S and RQSR-S (SEQ ID NO: 16) mutants (Figure 10E to Figure 10G). After purification, the average diameters of Omicron VLPs derived only from Omicron S protein were 105.5 ± 0.4 nm, 114.1 ± 1.0 nm, and 124.4 ± 0.5 nm for the original, 2P-S, and RQSR-2P-S mutants, respectively (Figure 10E to Figure 10G). This is the first evidence that overexpression of coronavirus S protein alone is sufficient to drive budding of intracellular coronavirus virions and efficient release of VLPs in the extracellular space and cell culture medium (Figure 10H).

Example 8 Omicron variant VLPs have reduced ability to elicit neutralizing antibodies. Vaccine candidates are evaluated by immunizing mice using a prime-plus (2 injections) vaccine schedule. VLPs derived from Delta and Omicron variants and mutants of S protein with AS03-like adjuvant (squalene-oil-in-water provided by RuenHuei Biopharmaceuticals Inc.) compared to 2P-S VLPs derived from ancestral S protein sequences. formulations) showed much lower immunogenicity. Therefore, increasing the dose of Omicron VLP to five times (containing 3.75 μg S protein/mouse) and formulating/not formulating the ancestral 2P-S VLP (containing 0.75 μg S protein/mouse) into a bivalent vaccine provided the best results for our studies in animals. Strategies for new or additional vaccines in experiments. Female K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice (n = 7 or 8, 8 weeks old) were treated with monovalent Omicron S VLP (wild-type BA.1 or its RQSR-2P mutant ), and their combination with ancestral 2P-S VLP (1:5 W/W) mixed with AS03-like adjuvant (1:1, V/V) as respective bivalent vaccines were administered subcutaneously on days 0 and 21 injection, and blood was drawn on day 35. In ELISA, the specific IgG potency of pooled antisera against Omicron-wt-S VLP reached GMT: 2 × 10 ⁵ . The neutralizing potency of the immune serum indicated by the 50% pseudovirus neutralizing potency (PVNT ₅₀ ) of O-RQSR-2P-S and the two bivalent vaccines against Omicron BA.1 pseudotyped lentivirus was significantly higher than that of O-wt -S. The neutralizing power of the bivalent vaccine (Omicron 2P-S and Omicron O-wt-S or O-RQSR-2P-S) against the PVNT ₅₀ of BA.5 in vaccinated mice was significantly higher than that of the monovalent group, indicating that Contribution due to cross-protective antibodies arising from the ancestral 2P-S VLP (Fig. 10I). In comparison, these VLPs formulated with MF59-like adjuvants produced lower neutralizing potency (data not shown). Our data suggest that the attenuated immunogenicity of Omicron VLPs (containing 3.75 μg S protein/mouse) requires formulation with more effective adjuvants, such as AS03-like squalene-oil-in-water emulsions or others, to stimulate high Neutralizing antibodies. Data indicate that VLPs presenting Omicron S with the RQSR-2P mutation can effectively constitute a strain-matched vaccine.

Example 9 H5N2 class Production of influenza virus particles.

capture allowed in humans FreeStyle 293F and insects High-Five The highest level of chromosomal gene loci expressing inducible foreign genes in cell lines

In order to produce VLPs of H5N2 influenza virus (A/duck/Taiwan/01006/2015/H5N2) using the human 293F cell line and the insect High-Five cell line, we constructed a construct designed to perform GFP in mammalian and insect expression systems. Doxycycline-inducible expression of plastids. In particular, the promoter driving expression in insect cells needs to be modified from the gene promoter of the Douglas-fir polyhedral multicapsid nuclear polyhedrosis virus (OpMNPV) immediate early 2 (IE2) to a strong doxycycline-inducible promoter and from The promoter of the actin A1 gene of the Cecropia moth was modified into a doxycycline-inducible weak promoter. In principle, the constitutively expressed Tet repressor inhibits the expression of IE2 and the Cecropia actin A1 promoter by binding to the tet operator 2 (TetO2) inserted downstream of the TATA box until the Tet repressor is activated by multiple Released by cerebrocycline treatment. We then prepared founder cells by stable transfection in FreeStyle 293F using FreeStyle MAX reagent. Cells exhibiting phenotypes of low basal and high induced GFP expression were twice enriched by flow cytometry-assisted cell sorting (FACS). Isolate individual cells from the enriched cells to obtain the best founder cell line.

Engineering transformation and carrying FRT Several transgenic gene cassette clusters are flanked, CMV/TO Promoter-driven and expressive 3 or 4 Donor plasmid for viral structural proteins

It is known that co-expression of S, M, E and/or N proteins in the same mammalian cells releases VLPs into the cell culture medium. Therefore, we constructed CMV/TO driven M-IRES-E, S, with/without N, distributed in 2 or 3 tandem genes in the same plastid. The entire transgenic gene cluster is adjacent to two FRT sites (F and Fn) upstream and downstream to become the target cassette of FLPe recombinase. The sequence of the H5 protein is shown as SEQ ID NO: 18; the sequence of the N2 protein is shown as SEQ ID NO: 20; the sequence of the M1 protein is shown as SEQ ID NO: 22); and the sequence of the M2 protein is shown as SEQ ID NO: 24.

via FLP Recombinase is co-transfected with a "gene exchange" donor cassette to achieve site-specific insertion of all transgenic genes.

Donor plasmids and FLPe expression plasmids were co-transfected into founder cell lines derived from human FreeStyle 293F and insect High-Five cell lines, respectively. Transfected cells were amplified using FACS and enriched by GFP deletion expression. Cells that showed re-sorting and enrichment were obtained by deleting GFP and HA in H5N2 influenza VLPs. A single cell line of the gene exchange cells was isolated, and the single cell line with the highest HA expression amount and background GFP expression amount after 2 days of doxycycline induction was screened. In the absence of doxycycline, HA should show low levels of staining. Using this "rapid multigene overexpression system", we generated stable VLP-producing 293F monoline cells.

H5N2 influenza VLP production and purification.

A stable VLP-producing single cell line (#71) was expanded to a density of 2 × ¹⁰ cells/ml in serum-free suspension cell culture in FreeStyle™ 293 Expression Medium. VLP expression and release are initiated by adding a working concentration of 1 µg/mL doxycycline to cell cultures. The doubling time of 293F cells in the healthy exponential growth phase is 22 to 28 hours. Cell density can be maintained at up to 3 × 10 ⁶ cells/ml in shaking or rotating cultures and up to 4 × 10 ⁶ cells/ml in bioreactor cultures. The medium was refreshed every 3 days, and VLPs were collected from the conditioned medium, filtered through 0.45 µm to remove cell debris and other large aggregates, concentrated by tangential flow filtration (TFF), and finally passed through a two-step sucrose gradient. (30% and then 40%-60%) ultracentrifugation or 1-step column chromatography Capto Core 700 Multimodal (MMC) and diafiltration, or 2-step column chromatography using Capto DeVirS and Capto Core 700 for purification , to eliminate most biological impurities.

The results are shown in Figures 11A to 11H. Successfully produced and purified H5N2 influenza VLPs.

Although the invention has been described in connection with the specific embodiments illustrated, many alternatives, modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are deemed to be within the scope of this invention.

Figures 1A to 1F show the production and characterization of 2P-S VLPs and D614G-S VLPs from 293F stable monoline cells. Figure 1A shows the pattern of spikes in 2P-S VLP and D614G-S VLP. TM, transmembrane domain; CT, cytoplasmic domain. Figure 1B shows immunofluorescence staining with anti-S1, anti-S2 antibodies and DAPI in the 293F stable cell line stably expressing different spikes of SARS-CoV-2. Figures 1C and 1D show that 2P-S VLPs (Figure 1C) and D614G-S VLPs (Figure 1D) were detected by anti-S1 and S2 antibodies in Western blotting. VLP samples were treated with no reduction and no boiling (N), no reduction and boiling (NB), and reduction and boiling (RB) before loading. Figure 1E shows physical analysis of VLPs determined by dynamic light scattering (DLS). Figure 1F shows central sections through cryo-electron microscopy tomography of 2P-S VLP and D614G-S VLP. The distribution of spikes can be observed on the surface of both VLPs. Scale bar: 100 nm.

Figures 2A to 2C show the immune response of VLPs in C57BL/6 mice. Figure 1A shows the vaccination protocol in the mouse model, including the immunization strategy. Figure 1B shows that D614G-S VLP was used as an antigen and detected in Western blotting using pooled antisera from mice immunized with different VLP vaccine formulations. N, non-reducing and non-boiling; NB, non-reducing and boiling; RB, reducing and boiling. Figure 1C shows spleen cells collected on day 14 after prime-boost vaccination and subsequently stimulated for 24 hours without or with D614G-S VLP containing 0.75 μg S. Expressed by the number of spots formed by cells capable of secreting IFN-γ or IL-4 per 5 × 10 ⁵ spleen cells. Bars, geometric mean; bars, 95% confidence interval (CI).

Figures 3A to 3D show immunization of VLPs in hamsters with AddaVax as adjuvant. Figure 3A shows the vaccination schedule of the hamster model, including the time points of immunization and viral challenge. Figure 3B shows the determination of the neutralizing potency of antisera from immunized hamsters at week 2 after the addition of a booster dose in a CPE-based colorimetric live virus microneutralization assay. Scatter plots represent individual data points and are overlaid with a horizontal line at the geometric mean, with 95% CI. Figure 3C shows the use of a pseudovirus carrying the S protein of SARS-CoV-2 to determine the neutralizing potency of immunized hamster antisera against the pseudovirus. Time plot with individual data points; line, geometric mean; bars, 95% CI. Figure 3D shows that D614G-S VLP can be recognized by VLP-immunized hamster pooled antisera in Western blotting. N, non-reducing and non-boiling; NB, non-reducing and non-boiling.

Figures 4A to 4F show the protective efficacy of VLP vaccines in immunized hamsters. Figure 4A shows the median body weight change in vaccinated hamsters after challenge. Points, median; bars, 95% CI. Figures 4B to 4D show the individual body weight changes and median values after challenge in hamsters vaccinated with PBS (Figure 4B), 2P-S (Figure 4C) and D614G-S (Figure 4D). Figure 4E shows the expression of the viral E gene in the lungs and duodenum of vaccinated hamsters at 3 dpi (left) and 6 dpi (right) measured by RT-PCR. The graph shows the geometric mean with 95% CI. Figure 4F shows that the TCID ₅₀ of SARS-CoV 2 in lung and duodenal tissue lysates at 3 dpi (left) and 6 dpi (right) was determined and represented as a scatter plot. Points, individual data; line, geometric mean; bars, 95% CI.

Figures 5A to 5G show histopathological analysis of vaccinated hamsters after SARS-CoV-2 infection. Figure 5A shows the pathological analysis of H&E staining. Scale bar, 500 μm. Figure 5B shows the quantification of the extent of lung parenchymal lesions in challenged hamsters at 3 dpi and 6 dpi, as a scatter plot. Points, individual data; line, geometric mean; bars, 95% CI. Figure 5C shows the determination of viral content in IHC using SARS-CoV-2-N antibodies. Scale bar, 100 μm. Figures 5D to 5G show the immune response of hamsters challenged with the virus. IHC analysis of IFN responses, neutrophils, macrophages, and CD3 T cells using MX1 (Figure 5D), MPO (Figure 5E), IBA-1 (Figure 5F), and CD3 (Figure 5G) antibodies, respectively. Examine lung pathology slide samples.

Figure 6A and Figure 6B show that 2P-S VLP (Figure 6A) and D614G-S VLP (Figure 6B) can be recognized by antisera from patients who recovered from COVID-19 infection.

Figures 7A and 7B show quantification of spike protein content in 2P-S and D614G-S VLPs using anti-S1 (Figure 7A) and anti-S2 (Figure 7B) antibodies in Western blotting.

Figures 8A and 8B show histopathological analysis of uninfected vaccinated hamsters. Figure 8A shows H&E staining. Figure 8B shows the immune response of hamsters challenged with the virus. IHC analysis of lung pathological section samples was performed using MX1, MPO, IBA-1 and CD3 antibodies to detect IFN response, neutrophils, macrophages and CD3 T cells respectively.

Figures 9A-9C show the production of VLPs derived from Delta variants of SARS-CoV-2. The repeated evolution of new mutant strains of SARS-CoV-2 that are becoming more and more infectious shows that as spike mutations accumulate, mutations that enhance adaptability follow. Therefore, we created a series of VLP production cell lines and followed the latest variants of high concern (VOC). Using our VLP production system in the 293F cell line, we recombinantly designed and transformed two series of VLPs based on the spike protein sequences of SARS-CoV-2 Delta and Omicron VOC. We have generated stably expressed production cell lines that co-express the S protein carrying the different mutations indicated, including prototype (or-) Delta, GSAS-2P and GSAS mutants, as well as M and E proteins, thereby producing Three different Delta-VLPs. The S protein in VLPs was qualitatively analyzed by immunoblotting using anti-S1 (Cat. No. 40592-T62, Sino Biological, Beijing, China) and anti-S2 antibodies (Cat. No. 40590-D001, Sino Biological). The spherical morphology of VLPs was observed by cryo-electron microtomography (ECT or cryoET), with corresponding different spikes on the surface.

Figures 10A to 10D show the production of VLPs derived from Omicron variants of SARS-CoV-2. Similarly, we produced Omicron VLPs by co-expressing Omicron S and M and E proteins carrying the different mutations indicated above. The S protein in Omicron VLP was qualitatively analyzed by immunoblotting using anti-S1 (Cat. No. 40592-MM117, Sino Biological) and anti-S2 antibodies (Cat. No. 40590-D001, Sino Biological). ETC shows that the spherical morphology of Omicron VLP exhibits different spikes. The average diameters of Omicron VLPs derived from S, M, and E proteins were 114.8 ± 0.8 nm, 109.3 ± 0.4 nm, and 110.7 ± 0.7 for the original, 2P-S, RQSR-2P-S, and GSAS-2P-S mutants, respectively. nm and 96.4 ± 0.4 nm.

Figures 10E to 10H demonstrate that overexpression of S protein is sufficient to drive VLP release from animal cells. We analyzed the proteome of VLPs derived from the 293F cell line that overexpressed the S, M, and E proteins of the original strain and found that the amounts of M and E proteins incorporated into the VLPs were almost undetectable. Therefore, we investigated whether overexpression of S protein alone could drive budding of VLPs out of transgenic cells. Importantly, VLPs can be produced and released in the culture medium only by overexpressing the S protein in 293F cell lines, including the original strain S (wild type) and its GSAS-2P-S mutant, Omicron S (prototype) and Omicron 2P -S and RQSR-S mutants (Figure 10E to Figure 10G). After purification, the average diameters of Omicron VLPs produced using only Omicron S protein were 105.5 ± 0.4 nm, 114.1 ± 1.0 nm, and 124.4 ± 0.5 nm for the prototype, 2P-S, and RQSR-2P-S mutants, respectively (Figure 10E to Figure 10G). This is unprecedented evidence that expression of the coronavirus S protein alone is sufficient to drive the budding of intracellular coronavirus-like particles (marked by arrows) and efficiently release VLPs in the extracellular space and cell culture medium (Figure 10H).

Figure 10I shows that despite poor immunogenicity, the VLP vaccine of the Omicron BA.1 variant can elicit neutralizing antibodies. The efficacy of vaccine candidates has been evaluated by immunizing with prime-plus doses (2 injections) of the same vaccine in a mouse model. Compared with 2P-S VLP derived from the original strain S protein sequence, VLP derived from Delta and Omicron VOC S protein and its mutants with MF59 adjuvant (1:1, V/V, provided by RuenHuei Biopharmaceuticals Inc. ) formulations exhibit very low immunogenicity. Therefore, we increased the dose of Omicron VLP to five times (containing 3.75 μg S protein/mouse) and incorporated the original strain of 2P-S VLP (containing 0.75 μg S protein) in animal experiments on new or additional vaccines. /mouse) into a bivalent vaccine formulation for comparison. Female K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice (n = 7 or 8, 8 weeks old) were treated with monovalent Omicron S VLP (prototype BA.1 or its RQSR-2P mutant) , and their combination with 2P-S VLP (1:5 W/W) of the original strain mixed with AS03 adjuvant (1:1, V/V, manufactured by RuenHuei Biopharmaceuticals Inc.) as their respective The bivalent vaccine was injected subcutaneously on days 0 and 21, and blood was drawn on day 35. In ELISA, the specific IgG potency of the pooled antisera against all vaccines containing Omicron VLP reached GMT: 2 × 10 ⁵ . The neutralizing potency of immune serum against Omicron BA.1 pseudotyped lentivirus, as shown by the 50% pseudovirus neutralizing potency (PVNT ₅₀ ) of O-RQSR-2P-S and the two bivalent vaccines, was significantly higher than that of O-or -S (Omicron prototype spike). The PVNT ₅₀ values (neutralizing potency) of mice immunized with the bivalent vaccine (2P-S plus Omicron O-or-S or O-RQSR-2P-S) against BA.5 were significantly higher than those based on BA.1 The monovalent group means that the neutralizing antibody response generated by Omicron VLP is quite specific to the same Omicron subvariant, unlike the original strain 2P-S VLP vaccine. Our data show that the immunogenicity of Omicron VLP is weakened, so the antigen dose needs to be increased (containing 3.75 μg S protein/mouse) and more powerful adjuvants (such as AS03 or others) need to be used to enhance and prolong the response by the target. Cellular and humoral immune responses stimulated by Omicron and future VOC VLP vaccines. Experimental data show that VLPs expressing Omicron S with RQSR-2P mutations can effectively constitute a vaccine matching this virus strain.

Figures 11A to 11H show the establishment of a stable 293F cell line producing H5N2-VLP and the characterization of H5N2-VLP. Figure 11A shows in step I that the human 293F cell line was stably transfected with a GFP reporter plasmid to capture highly expressed gene loci in the gene body. After a single cell line is isolated, a single cell line with a single set of reporters is used as the founder cell line for qualitative analysis. In step II, the founder cell line is then co-transfected with FLPe and donor plastids to exchange GFP with the H5N2-VLP gene cluster. Figure 11B shows that after doxycycline induction, gene-swap cells are enriched based on GFP deficiency. Figure 11C shows that individual cells were isolated and qualitatively analyzed for the inducible expression of their VLP genes. Figure 11D shows that VLP-producing cells were scaled up in suspension culture and induced by adding 1 µg/mL doxycycline to the culture medium. H5N2-VLPs were harvested from conditioned medium and purified by sucrose density gradient ultracentrifugation. The purified H5N2-VLPs (Batch 1 and Batch 2) were analyzed for hemagglutination activity. Figure 11E shows that purified H5N2-VLP was negatively stained with 2% uranyl acetate and observed by transmission electron microscopy (TEM) at 100,000× magnification. Figure 11F shows that the particle size and distribution of purified H5N2-VLP were determined and analyzed by dynamic light scattering (DLS). Figure 11G shows measurement of NA activity by NA-star influenza neuraminidase inhibitor resistance detection kit (Thermo Fisher). Provides 150,000 RLU/sec NA activity per μg of H5N2-VLP. Figure 11H shows analysis of the HA protein of purified H5N2-VLPs by Western blotting using anti-H5 antibodies.

Figures 12A to 12E show the establishment of a stable insect High-Five cell line for producing H5N2-VLP and the qualitative analysis of H5N2-VLP. Figure 12A shows that the insect High-5 cell line was stably transfected with a GFP reporter plasmid to capture highly expressed gene loci in its gene body. The founder cell line was subsequently co-transfected with insect promoter-driven FLPe and donor plastid to exchange GFP with the H5N2-VLP gene cluster. After doxycycline induction, cells that completed gene exchange were enriched by loss of GFP. Figure 12B shows that a single cell line was isolated and qualitatively analyzed against a single cell line with a single set of reporters as the founder cell line. GFP images of representative founder cell lines. Figure 12C shows that a single cell line was isolated and the inducible expression of its VLP gene was qualitatively analyzed by qRT-PCR. Figure 12D shows the NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kit (Thermo Fisher) measured the neuraminidase activity of VLP-producing cells. Figure 12E shows that purified H5N2-VLP was negatively stained by 2% uranyl acetate and observed by transmission electron microscopy (TEM) at 200,000× magnification.

Figure 13 shows the targeting cassette for mammalian cell systems.

Figure 14 shows the target cassette for the insect cell system.

Figure 15 shows the map of pGEMT-RMCE1-CMVto-sfGFP.

Figure 16 shows the map of pUC57.Insect RMCR1.

TW202334433A_111134790_SEQL.xml

Claims (29)

An animal cell stably expressing virus-like particles (VLP) contains an inducible expression cassette for site-directed recombination of one or more VLP genes. The animal cell of claim 1, wherein the animal cell is an insect cell or a mammalian cell. The animal cell of claim 1, wherein the mammalian cell is a human cell. Such as the animal cell of claim 1, wherein the virus particle contains a coronavirus structural protein or an influenza virus structural protein. Such as the animal cell of claim 4, wherein the coronavirus structural protein is the structural protein of SARS-CoV-2 (COVID-19). For example, the animal cell of claim 5, wherein the SARS-CoV-2 is a Delta and Omicron variant of SARS-CoV-2. Such as the animal cell of claim 4, wherein the coronavirus structural protein includes spike protein (S), membrane protein (M) and envelope protein (E). Such as the animal cell of claim 7, wherein the spike protein is native D614G spike protein (SEQ ID NO: 6), diproline mutant spike protein (2P-S) (SEQ ID NO: 8), D614G- S mutant spike protein, Delta-or spike protein (SEQ ID NO: 10), Delta-GSAS 2P mutant spike protein (SEQ ID NO: 12), Omicron-or spike protein (SEQ ID NO: 14) or Omicron-GSAS 2P spike protein (SEQ ID NO: 16). Such as the animal cell of claim 8, wherein the spike protein is encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 7, 9, 11, 13, 15 and 17. Such as the animal cell of claim 4, wherein the influenza virus structural protein is the structural protein of H5N2 or H3N2 influenza virus. Such as the animal cell of claim 4, wherein the influenza virus structural protein includes hemagglutinin (HA), neuraminidase (NA) and matrix proteins (M1 and M2). Such as the animal cell of claim 4, wherein the influenza virus structural protein is selected from the group consisting of: H5 protein (SEQ ID NO: 18), N2 protein (SEQ ID NO: 20), M1 protein (SEQ ID NO: 22 ) and M2 protein (SEQ ID NO: 24). Such as the animal cell of claim 12, wherein the influenza virus structural protein is encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 19, 21, 23 and 25. Such as the animal cells of claim 1, which are stably transfected with a target cassette containing an inducible tetracycline-inducible promoter-reporter or a doxycycline-inducible promoter-reporter of the Flp/FRT recombination system and stabilized The expressed tetracycline repressor cassette; and the target cassette and the tetracycline repressor cassette are genetically exchanged through co-transfection with FLPe recombinase to achieve site-specific insertion of all VLP genes. The animal cell of claim 1, wherein the inducible expression cassette includes a tetracycline-inducible promoter or a doxycycline-inducible promoter. For example, the animal cells of claim 1 stably express the tetracycline inhibitor cassette. The animal cell of claim 16, wherein the tetracycline suppressor cassette includes a tetracycline suppressor gene and a paimimycin S resistance gene linked by a self-cleaving 2A peptide derived from porcine Tiesca virus 1. Such as the animal cell of claim 1, wherein the inducible expression cassette includes CMV/TO, Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) immediate early 2 (IE2) and Antheraea pernyl Actin A1 promoter. Such as the animal cell of claim 16, wherein the stable expression cassette is an EF1a/eIF4g-pCI-TetR-P2A-BSD cassette. A method for producing virus-like particles, comprising culturing the animal cells of any one of claims 1 to 19 and harvesting the virus-like particles. A virus-like particle produced by the method of claim 20. A vaccine composition comprising an immunologically effective amount of viral particles as claimed in claim 21. The vaccine composition of claim 22, further comprising an adjuvant. A use of the vaccine composition of claim 22 for the manufacture of medicaments for preventing viral infection in individuals. Such as requesting the use of item 24, wherein the viral infection is coronavirus infection or influenza virus infection. Such as the use of request item 24, wherein the viral infection is SARS-CoV-2 infection or H5N2 or H3N2 influenza virus infection. Such as the use of claim 24, wherein the drug is used to prevent virus replication or relieve symptoms caused by the virus infection. A use of the vaccine composition of claim 22 for the manufacture of medicaments for individuals to produce antibodies specific to the VLPs. The use of claim 28, wherein the antibodies specific for the VLPs are further harvested from the individual.